Systematic evolution of ligands by exponential enrichment: chimeric selex

Abstract

Methods are disclosed for producing chimeric nucleic acid molecules with two or more functions. A chimeric library is generated in which individual chimeric molecules combine the functions or characteristics of two or more parent libraries, each parent library having been selected through the SELEX procedure for a specific function or feature. The chimeric molecules of this invention are useful in a variety of ways, including having improved affinities for a target molecule, enhancing assembly of multi-component molecules, and promoting reactions between two molecules.

We claim: 
     
       1. A method of identifying chimeric nucleic acid ligands having high binding affinity for first and second target molecules from a chimeric nucleic acid ligand library, said method comprising: a) preparing a chimeric nucleic acid ligand library generated according to a method comprising: i) generating a first library of nucleic acid ligands selected through the SELEX procedure for binding to said first target molecule, said nucleic acid ligands having a 3' fixed sequence, and producing the double-stranded form of said first library of nucleic acid ligands;   ii) generating a second library of nucleic acid ligands selected through the SELEX procedure for binding to said second target molecule, said nucleic acid ligands having a 5' fixed sequence identical to the 3' fixed sequence of the nucleic acids of said first library, and producing the double-stranded form of said second library of nucleic acid ligands;   iii) mixing said first and second libraries under conditions which promote interlibrary annealing;   iv) forming chimeric nucleic acid ligands by enzymatically extending the recessed 3' ends while copying the 5' extensions of each annealed pair, to yield a double-stranded chimeric nucleic acid ligand library;   v) amplifying said double stranded nucleic acid ligand library chimeric;     b) contacting said amplified chimeric nucleic acid ligand library with said first and second target molecules, wherein chimeric nucleic acid ligands having an increased affinity to said first and second target molecules may be partitioned from the remainder of the chimeric nucleic acid ligand library;   c) partitioning said increased affinity chimeric nucleic acid ligands from the remainder of said chimeric nucleic acid ligand library; and   d) amplifying said increased affinity chimeric nucleic acid ligands to yield a mixture of chimeric nucleic acid ligands enriched in members which bind to said first and second target molecules, whereby chimeric nucleic acid ligands having high binding affinity to said first and second target molecules are identified.   
     
     
       2. A method of identifying chimeric nucleic acid ligands having high binding affinity for first and second target molecules from a chimeric nucleic acid ligand library, said method comprising: a) preparing a chimeric nucleic acid ligand library generated according to a method comprising: i) generating a first library of nucleic acid ligands selected through the SELEX procedure which bind to said first target molecules, said nucleic acid ligands having a 3' fixed sequence and a 5'-monophosphate;   ii) generating a second library of nucleic acid ligands selected through the SELEX procedure which bind to said second target molecule; and   iii) mixing said first and second libraries under conditions wherein the nucleic acid ligands of the first library are enzymatically or chemically joined to the nucleic acids of the second library to form chimeric nucleic acid ligands;   iv) amplifying said chimeric nucleic acid ligands, to yield said chimeric nucleic acid ligand library;     b) contacting said amplified chimeric nucleic acid ligand library with said first and second target molecules, wherein chimeric nucleic acid ligands having an increased affinity to said first and second target molecules may be partitioned from the remainder of the chimeric nucleic acid ligand library;   c) partitioning said increased affinity chimeric nucleic acid ligands from the remainder of said chimeric nucleic acid ligand library; and   d) amplifying said increased affinity chimeric nucleic acid ligands to yield a mixture of chimeric nucleic acid ligands enriched in members which bind to said first and second target molecules, whereby chimeric nucleic acid ligands having high binding affinity to said first and second target molecules are identified.   
     
     
       3. A method of identifying chimeric nucleic acid ligands having high binding affinity for a target molecule from a chimeric nucleic acid ligand library, said method comprising: a) preparing a chimeric nucleic acid ligand library generated according to a method comprising: i) generating a first library of nucleic acid ligands selected through the SELEX procedure for binding to said target molecule wherein each of said nucleic acid ligands is modified at the 3' end to facilitate linking of first and second members of said first library with each other; and   ii) chemically reacting said first and second members of said first library of nucleic acid ligands to form chimeric 3'-3'-linked nucleic acid ligands which comprise said chimeric nucleic acid library;     b) contacting said chimeric nucleic acid ligand library with said target molecule, wherein chimeric nucleic acid ligands having an increased affinity to said target molecule may be partitioned from the remainder of the chimeric nucleic acid ligand library;   c) partitioning said increased affinity chimeric nucleic acid ligands from the remainder of said chimeric nucleic acid ligand library; and   d) amplifying said first and second members of said increased affinity chimeric nucleic acid ligands, to yield a mixture of nucleic acid ligand members, enriched in nucleic acid ligand members which bind to said target molecule;   e) chemically reacting said mixture of amplified nucleic acid ligand members to form increased affinity chimeric 3'-3'-linked nucleic acid ligands, whereby chimeric nucleic acid molecules having high binding affinity to said target molecule are identified.   
     
     
       4. The method of claim 1, wherein said chimeric nucleic acid ligand library is generated according to a method further comprising between step v) and step b), the step: transcribing said amplified double-stranded chimeric nucleic acid ligand library to yield a single-stranded RNA chimeric nucleic acid ligand library.   
     
     
       5. The method of claim 1, wherein said chimeric nucleic acid ligand library is generated according to a method further comprising between step v) and step b), the step: vi) separating the strands of said amplified double-stranded chimeric nucleic acid ligand library to yield a single-stranded DNA chimeric nucleic acid ligand library.